scholarly journals Multigenic engineering of the chloroplast genome in the green alga Chlamydomonas reinhardtii

Microbiology ◽  
2020 ◽  
Vol 166 (6) ◽  
pp. 510-515 ◽  
Author(s):  
Marco Larrea-Alvarez ◽  
Saul Purton
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Chloroplast Genome ◽  
Metabolic Pathways ◽  
Short Communication ◽  
Selectable Marker ◽  
Gene Products ◽  
Proof Of Concept ◽  
Transgenic Lines ◽  
Almost All ◽  
Algal Chloroplast

The chloroplast of microalgae such as Chlamydomonas reinhardtii represents an attractive chassis for light-driven production of novel recombinant proteins and metabolites. Methods for the introduction and expression of transgenes in the chloroplast genome (=plastome) of C. reinhardtii are well-established and over 100 different proteins have been successfully produced. However, in almost all reported cases the complexity of the genetic engineering is low, and typically involves introduction into the plastome of just a single transgene together with a selectable marker. In order to exploit fully the potential of the algal chassis it is necessary to establish methods for multigenic engineering in which many transgenes can be stably incorporated into the plastome. This would allow the synthesis of multi-subunit proteins and the introduction into the chloroplast of whole new metabolic pathways. In this short communication we report a proof-of-concept study involving both a combinatorial and serial approach, with the goal of synthesizing five different test proteins in the C. reinhardtii chloroplast. Analysis of the various transgenic lines confirmed the successful integration of the transgenes and accumulation of the gene products. However, the work also highlights an issue of genetic instability when using the same untranslated region for each of the transgenes. Our findings therefore help to define appropriate strategies for robust multigenic engineering of the algal chloroplast.

Recombinant Protein Production in Microalgae: Emerging Trends

2020 ◽  
Vol 27 (2) ◽  
pp. 105-110 ◽  
Author(s):  
Niaz Ahmad ◽  
Muhammad Aamer Mehmood ◽  
Sana Malik
Keyword(s):  
Chloroplast Genome ◽  
Recombinant Proteins ◽  
Large Scale ◽  
Higher Plants ◽  
Scale Up ◽  
Chloroplast Transformation ◽  
Point Of View ◽  
Nuclear Transformation ◽  
Scale Production ◽  
Algal Chloroplast

: In recent years, microalgae have emerged as an alternative platform for large-scale production of recombinant proteins for different commercial applications. As a production platform, it has several advantages, including rapid growth, easily scale up and ability to grow with or without the external carbon source. Genetic transformation of several species has been established. Of these, Chlamydomonas reinhardtii has become significantly attractive for its potential to express foreign proteins inexpensively. All its three genomes – nuclear, mitochondrial and chloroplastic – have been sequenced. As a result, a wealth of information about its genetic machinery, protein expression mechanism (transcription, translation and post-translational modifications) is available. Over the years, various molecular tools have been developed for the manipulation of all these genomes. Various studies show that the transformation of the chloroplast genome has several advantages over nuclear transformation from the biopharming point of view. According to a recent survey, over 100 recombinant proteins have been expressed in algal chloroplasts. However, the expression levels achieved in the algal chloroplast genome are generally lower compared to the chloroplasts of higher plants. Work is therefore needed to make the algal chloroplast transformation commercially competitive. In this review, we discuss some examples from the algal research, which could play their role in making algal chloroplast commercially successful.

scholarly journals Recent Advances of Microbiome-Associated Metabolomics Profiling in Liver Disease: Principles, Mechanisms, and Applications

2021 ◽  
Vol 22 (3) ◽  
pp. 1160
Author(s):  
Ganesan Raja ◽  
Haripriya Gupta ◽  
Yoseph Asmelash Gebru ◽  
Gi Soo Youn ◽  
Ye Rin Choi ◽  
...  
Keyword(s):  
Liver Disease ◽  
Metabolic Pathways ◽  
High Throughput Screening ◽  
Proof Of Concept ◽  
Metabolic Factors ◽  
Metabolomics Data ◽  
Liver Health ◽  
Activity Screening ◽  
Cellular Microenvironments ◽  
Endogenous Metabolites

Advances in high-throughput screening of metabolic stability in liver and gut microbiota are able to identify and quantify small-molecule metabolites (metabolome) in different cellular microenvironments that are closest to their phenotypes. Metagenomics and metabolomics are largely recognized to be the “-omics” disciplines for clinical therapeutic screening. Here, metabolomics activity screening in liver disease (LD) and gut microbiomes has significantly delivered the integration of metabolomics data (i.e., a set of endogenous metabolites) with metabolic pathways in cellular environments that can be tested for biological functions (i.e., phenotypes). A growing literature in LD and gut microbiomes reports the use of metabolites as therapeutic targets or biomarkers. Although growing evidence connects liver fibrosis, cirrhosis, and hepatocellular carcinoma, the genetic and metabolic factors are still mainly unknown. Herein, we reviewed proof-of-concept mechanisms for metabolomics-based LD and gut microbiotas’ role from several studies (nuclear magnetic resonance, gas/lipid chromatography, spectroscopy coupled with mass spectrometry, and capillary electrophoresis). A deeper understanding of these axes is a prerequisite for optimizing therapeutic strategies to improve liver health.

scholarly journals A Comprehensive Understanding of Electro-Fermentation

Fermentation ◽  
2020 ◽  
Vol 6 (3) ◽  
pp. 92 ◽  
Author(s):  
Drishti Dinesh Bhagchandanii ◽  
Rishi Pramod Babu ◽  
Jayesh M. Sonawane ◽  
Namita Khanna ◽  
Soumya Pandit ◽  
...  
Keyword(s):  
Metabolic Pathways ◽  
Biomass Yield ◽  
Carbon Chain ◽  
Industrial Applications ◽  
Chain Elongation ◽  
Comprehensive Understanding ◽  
Specific Product ◽  
Microbial Strains ◽  
Metabolic Reactions ◽  
Almost All

Electro-fermentation (EF) is an upcoming technology that can control the metabolism of exoelectrogenic bacteria (i.e., bacteria that transfer electrons using an extracellular mechanism). The fermenter consists of electrodes that act as sink and source for the production and movement of electrons and protons, thus generating electricity and producing valuable products. The conventional process of fermentation has several drawbacks that restrict their application and economic viability. Additionally, metabolic reactions taking place in traditional fermenters are often redox imbalanced. Almost all metabolic pathways and microbial strains have been studied, and EF can electrochemically control this. The process of EF can be used to optimize metabolic processes taking place in the fermenter by controlling the redox and pH imbalances and by stimulating carbon chain elongation or breakdown to improve the overall biomass yield and support the production of a specific product. This review briefly discusses microbe-electrode interactions, electro-fermenter designs, mixed-culture EF, and pure culture EF in industrial applications, electro methanogenesis, and the various products that could be hence generated using this process.

scholarly journals Endogenous Fluctuations of DNA Topology in the Chloroplast of Chlamydomonas reinhardtii

1998 ◽  
Vol 18 (12) ◽  
pp. 7235-7242 ◽  
Author(s):  
Maria L. Salvador ◽  
Uwe Klein ◽  
Lawrence Bogorad
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Chloroplast Genome ◽  
Continuous Light ◽  
Dna Supercoiling ◽  
Dna Topology ◽  
Chloroplast Gene ◽  
Chloroplast Gene Expression ◽  
Endogenous Fluctuations ◽  
Unicellular Green Alga ◽  
In Cells

ABSTRACT DNA supercoiling in the chloroplast of the unicellular green algaChlamydomonas reinhardtii was found to change with a diurnal rhythm in cells growing in alternating 12-h dark–12-h light periods. Highest and lowest DNA superhelicities occurred at the beginning and towards the end of the 12-h light periods, respectively. The fluctuations in DNA supercoiling occurred concurrently and in the same direction in two separate parts of the chloroplast genome, one containing the genes psaB, rbcL, andatpA and the other containing the atpB gene. Fluctuations were not confined to transcribed DNA regions, indicating simultaneous changes in DNA conformation all over the chloroplast genome. Because the diurnal fluctuations persisted in cells kept in continuous light, DNA supercoiling is judged to be under endogenous control. The endogenous fluctuations in chloroplast DNA topology correlated tightly with the endogenous fluctuations of overall chloroplast gene transcription and with those of the pool sizes of most chloroplast transcripts analyzed. This result suggests that DNA superhelical changes have a role in the regulation of chloroplast gene expression in Chlamydomonas.

The CRY1 gene in Chlamydomonas reinhardtii: structure and use as a dominant selectable marker for nuclear transformation

1994 ◽  
Vol 14 (6) ◽  
pp. 4011-4019
Author(s):  
J A Nelson ◽  
P B Savereide ◽  
P A Lefebvre
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Selectable Marker ◽  
Marker Gene ◽  
Small Subunit ◽  
Direct Selection ◽  
Nuclear Transformation ◽  
Ribulose Bisphosphate Carboxylase ◽  
Bisphosphate Carboxylase ◽  
Dominant Selectable Marker ◽  
Cry1 Gene

We have cloned and sequenced the CRY1 gene, encoding ribosomal protein S14 in Chlamydomonas reinhardtii, and found that it is highly similar to S14/rp59 proteins from other organisms, including mammals, Drosophila melanogaster, and Saccharomyces cerevisiae. We isolated a mutant strain resistant to the eukaryotic translational inhibitors cryptopleurine and emetine in which the resistance was due to a missense mutation (CRY1-1) in the CRY1 gene; resistance was dominant in heterozygous stable diploids. Cotransformation experiments using the CRY1-1 gene and the gene for nitrate reductase (NIT1) produced a low level of resistance to cryptopleurine and emetine. Resistance levels were increased when the CRY1-1 gene was placed under the control of a constitutive promoter from the ribulose bisphosphate carboxylase/oxygenase small subunit 2 (RBCS2) gene. We also found that the 5' untranslated region of the CRY1 gene was required for expression of the CRY1-1 transgene. Direct selection of emetine-resistant transformants was possible when transformed cells were first induced to differentiate into gametes by nitrogen starvation and then allowed to dedifferentiate back to vegetative cells before emetine selection was applied. With this transformation protocol, the RBCS2/CRY1-1 dominant selectable marker gene is a powerful tool for many molecular genetic applications in C. reinhardtii.

scholarly journals cAMP metabolism controls caspase-11 inflammasome activation and pyroptosis in sepsis

2019 ◽  
Vol 5 (5) ◽  
pp. eaav5562 ◽  
Author(s):  
Ruochan Chen ◽  
Ling Zeng ◽  
Shan Zhu ◽  
Jiao Liu ◽  
Herbert J. Zeh ◽  
...  
Keyword(s):  
Metabolic Pathways ◽  
Inflammatory Responses ◽  
Poly I:C ◽  
Inflammasome Activation ◽  
Proof Of Concept ◽  
Potential Therapeutic Target ◽  
Camp Metabolism ◽  
Camp Hydrolysis ◽  
Insight Into

The ability of cytosolic lipopolysaccharide (LPS) to activate caspase-11–dependent nonclassical inflammasome is intricately controlled to avoid excessive inflammatory responses. However, very little is known about the regulatory role of various metabolic pathways in the control of caspase-11 activation. Here, we demonstrate that l-adrenaline can act on receptor ADRA2B to inhibit the activation of the caspase-11 inflammasome by cytosolic LPS or Escherichia coli infection in macrophages. l-adrenaline–induced cAMP production via the enzyme ADCY4 promotes protein kinase A (PKA) activation, which then blocks the caspase-11–mediated proteolytic maturation of interleukin-1β, gasdermin D (GSDMD) cleavage, and consequent DAMP release. Inhibition of PDE8A-mediated cAMP hydrolysis limits caspase-11 inflammasome activation and pyroptosis in macrophages. Consequently, pharmacological modulation of the ADRA2B-ADCY4-PDE8A-PKA axis, knockout of caspase-11 (Casp11−/−), or Gsdmd inactivation (GsdmdI105N/I105N) similarly protects against LPS-induced lethality in poly(I:C)-primed mice. Our results provide previously unidentified mechanistic insight into immune regulation by cAMP and represent a proof of concept that immunometabolism constitutes a potential therapeutic target in sepsis.

scholarly journals Effects of microcompartmentation on flux distribution and metabolic pools in Chlamydomonas reinhardtii chloroplasts

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Anika Küken ◽  
Frederik Sommer ◽  
Liliya Yaneva-Roder ◽  
Luke CM Mackinder ◽  
Melanie Höhne ◽  
...  
Keyword(s):  
Experimental Data ◽  
Chlamydomonas Reinhardtii ◽  
Metabolic Pathways ◽  
Metabolic Flux ◽  
Flux Distribution ◽  
Photosynthetic Performance ◽  
Computational Approach ◽  
Cellular Processes ◽  
Diffusional Barrier ◽  
Computational Strategy

Cells and organelles are not homogeneous but include microcompartments that alter the spatiotemporal characteristics of cellular processes. The effects of microcompartmentation on metabolic pathways are however difficult to study experimentally. The pyrenoid is a microcompartment that is essential for a carbon concentrating mechanism (CCM) that improves the photosynthetic performance of eukaryotic algae. Using Chlamydomonas reinhardtii, we obtained experimental data on photosynthesis, metabolites, and proteins in CCM-induced and CCM-suppressed cells. We then employed a computational strategy to estimate how fluxes through the Calvin-Benson cycle are compartmented between the pyrenoid and the stroma. Our model predicts that ribulose-1,5-bisphosphate (RuBP), the substrate of Rubisco, and 3-phosphoglycerate (3PGA), its product, diffuse in and out of the pyrenoid, respectively, with higher fluxes in CCM-induced cells. It also indicates that there is no major diffusional barrier to metabolic flux between the pyrenoid and stroma. Our computational approach represents a stepping stone to understanding microcompartmentalized CCM in other organisms.

Host-mediated RNAi of a Notch-like receptor gene inMeloidogyne incognitainduces nematode resistance

Parasitology ◽  
2018 ◽  
Vol 145 (14) ◽  
pp. 1896-1906
Author(s):  
Deshika Kohli ◽  
Parameswaran Chidambaranathan ◽  
J. Prasanth Tej Kumar ◽  
Ashish Kumar Singh ◽  
Anil Kumar ◽  
...  
Keyword(s):  
Transmembrane Domain ◽  
Receptor Gene ◽  
Nematode Resistance ◽  
Receptor Protein ◽  
Binding Motifs ◽  
C Elegans ◽  
Transgenic Lines ◽  
Germline Proliferation ◽  
Epidermal Growth ◽  
Almost All

AbstractGLP-1 (abnormal germline proliferation) is a Notch-like receptor protein that plays an essential role in pharyngeal development. In this study, an orthologue ofCaenorhabditis elegans glp-1was identified inMeloidogyne incognita. A computational analysis revealed that the orthologue contained almost all the domains present in theC. elegansgene: specifically, the LIN-12/Notch repeat, the ankyrin repeat, a transmembrane domain and different ligand-binding motifs were present in orthologue, but the epidermal growth factor-like motif was not observed. An expression analysis showed differential expression ofglp-1throughout the life cycle ofM. incognita, with relatively higher expression in the egg stage. To evaluate the silencing efficacy ofMi-glp-1, transgenicArabidopsisplants carrying double-stranded RNA constructs ofglp-1were generated, and infection of these plants withM. incognitaresulted in a 47–50% reduction in the numbers of galls, females and egg masses. Females obtained from the transgenic RNAi lines exhibited 40–60% reductions in the transcript levels of the targetedglp-1gene compared with females isolated from the control plants. Second-generation juveniles (J2s), which were descendants of the infected females from the transgenic lines, showed aberrant phenotypes. These J2s exhibited a significant decrease in the overall distance from the stylet to the metacorpus region, and this effect was accompanied by disruption around the metacorporeal bulb of the pharynx. The present study suggests a role for this gene in organ (pharynx) development during embryogenesis inM. incognitaand its potential use as a target in the management of nematode infestations in plants.

scholarly journals Blasticidin S Deaminase: A New Efficient Selectable Marker for Chlamydomonas reinhardtii

2020 ◽  
Vol 11 ◽  
Author(s):  
Félix de Carpentier ◽  
Jeanne Le Peillet ◽  
Nicolas D. Boisset ◽  
Pierre Crozet ◽  
Stéphane D. Lemaire ◽  
...  
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Selectable Marker ◽  
Blasticidin S

scholarly journals Synthetic glycolate metabolism pathways stimulate crop growth and productivity in the field

Science ◽  
2019 ◽  
Vol 363 (6422) ◽  
pp. eaat9077 ◽  
Author(s):  
Paul F. South ◽  
Amanda P. Cavanagh ◽  
Helen W. Liu ◽  
Donald R. Ort
Keyword(s):  
Quantum Yield ◽  
Crop Yield ◽  
Metabolic Pathways ◽  
Biomass Productivity ◽  
Field Trials ◽  
Agricultural Field ◽  
Bisphosphate Carboxylase ◽  
Transgenic Lines ◽  
Glycolate Metabolism ◽  
Pathway Flux

Photorespiration is required in C3 plants to metabolize toxic glycolate formed when ribulose-1,5-bisphosphate carboxylase-oxygenase oxygenates rather than carboxylates ribulose-1,5-bisphosphate. Depending on growing temperatures, photorespiration can reduce yields by 20 to 50% in C3 crops. Inspired by earlier work, we installed into tobacco chloroplasts synthetic glycolate metabolic pathways that are thought to be more efficient than the native pathway. Flux through the synthetic pathways was maximized by inhibiting glycolate export from the chloroplast. The synthetic pathways tested improved photosynthetic quantum yield by 20%. Numerous homozygous transgenic lines increased biomass productivity between 19 and 37% in replicated field trials. These results show that engineering alternative glycolate metabolic pathways into crop chloroplasts while inhibiting glycolate export into the native pathway can drive increases in C3 crop yield under agricultural field conditions.

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